Novel imaging composition and uses thereof

ABSTRACT

The invention discussed in this application relates to hydroxamic acid-based compounds that are useful as imaging agents when bound to an appropriate metal centre, particularly for the imaging of tumours.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/963,599, filed Apr. 26, 2018, which is a Continuation of U.S. patentapplication Ser. No. 15/518,333, filed Apr. 11, 2017, now U.S. Pat. No.9,980,930, issued on May 29, 2018 and is a National Stage Application,filed under 35 U.S.C. 371, of International Application No.PCT/AU2015/050640, filed on Oct. 16, 2015, which claims priority to, andthe benefit of, AU Application No. 2014904138, filed Oct. 16, 2014. Thecontents of each of these applications are incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to hydroxamic acid-based compounds thatare useful as imaging agents when bound to an appropriate metal centre,particularly for the imaging of tumours. The present invention alsorelates to compositions including the compounds, and to methods ofimaging patients using the compounds.

BACKGROUND OF THE INVENTION

Zirconium-89 (⁸⁹Zr) is a positron-emitting radionuclide that is used inmedical imaging applications. In particular, it is used in positronemission tomography (PET) for cancer detection and imaging. It has alonger half-life (t_(1/2)=79.3 hours) than other radionuclides used formedical imaging, such as ¹⁸F. For example, ¹⁸F has a t_(1/2) of 110minutes, which means that its use requires close proximity to acyclotron facility and rapid and high-yielding synthesis techniques forthe preparation of the agents into which it is incorporated. ⁸⁹Zr is notplagued by these same problems, which makes ⁸⁹Zr particularly attractivefor use in medical imaging applications.

Desferrioxamine (DFO) is a bacterial siderophore that has been usedsince the late 1960s to treat iron overload. The three hydroxamic acidgroups in DFO form co-ordination bonds with Fe³⁺ ions, essentiallymaking DFO a hexadentate ligand that chelates the Fe³⁺ ions. Due to theco-ordination geometry of ⁸⁹Zr, DFO has also been used as a chelator for⁸⁹Zr in PET imaging applications (Holland, J. P. et al (2012) Nature10:1586).

Other DFO-based radioisotope chelators have also been prepared for usein PET imaging applications. These includeN-succinyl-desferrioxamine-tetrafluorophenol ester (N-suc-DFO-TFP ester)p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS, also known asDFO-Ph-NCS) and desferrioxamine-maleimide (DFO-maleimide). All of thesechelators can be conjugated with antibodies or antibody fragments toprovide a means of targeting the imaging agent to the tumour to beimaged.

However, these chelators suffer from a number of disadvantages. Thesynthesis of N-suc-DFO-TFP ester involves the addition of Fe³⁺, toprevent the reaction of the tetrafluorophenol ester with one of thehydroxamate groups of desferrioxamine (DFO). Upon completion of thesynthesis (which includes the step of coupling N-suc-DFO-TFP ester to anantibody), the Fe³⁺ then needs to be removed. This is achieved using a100-fold molar excess of EDTA at a pH of 4.2-4.5. These conditions canbe detrimental to pH-sensitive antibodies.

With regard to DFO-Bz-NCS, if this compound is added to an antibodysolution too quickly without shaking or proper mixing, DFO-Bz-NCS causesthe formation of antibody aggregates. In addition, the stability of theradiolabelled and antibody-conjugated chelators is a concern when storedfor extended periods of time, and buffers containing chloride ions needto be avoided as they result in detachment of the radionuclide from thecomplex.

DFO-maleimide conjugates to antibodies via Michael addition to thiolgroups. There are two main issues with this. The first is that Michaeladditions to thiols can lead to mixtures of isomers. This is adisadvantage because the isomers may interact in different ways withbiological systems. The second issue is that Michael addition to thiolgroups is reversible. This increases the risk that theDFO-maleimide-radionulide complex will dissociate from the antibody,resulting in distribution of the complex throughout the body. This notonly decreases the imaging selectivity but also increases the likelihoodof toxic side effects as the radiation emitted from the radionuclide inthe complex interacts with other organs.

Therefore, there is a need to develop new agents for use withradioisotopes, which do not have these drawbacks.

Reference to any prior art in the specification is not an acknowledgmentor suggestion that this prior art forms part of the common generalknowledge in any jurisdiction or that this prior art could reasonably beexpected to be understood, regarded as relevant, and/or combined withother pieces of prior art by a skilled person in the art.

SUMMARY OF THE INVENTION

The present inventors have found that the compound of formula (I) setout below (also referred to herein as “DFO-squaramide” or “DFOSq”), andits conjugate with a biological molecule (when complexed to aradionuclide such as ⁸⁹Zr), is an effective PET imaging agent:

wherein L is a leaving group. In one embodiment, L is OR. R may beselected from C₁ to C₁₀ alkyl, C₁ to C₁₀ heteroalkyl, C₂ to C₁₀ alkene,C₂ to C₁₀ alkyne, and aryl, each of which is optionally substituted. Rmay be C₁ to C₁₀ alkyl (e.g. C₁ to C₆ alkyl, such as methyl, ethyl,propyl or butyl). R may be methyl or ethyl. R may be ethyl.

Therefore, in one aspect, the present invention relates to a compound offormula (I), as defined above, or a pharmaceutically acceptable saltthereof.

In another aspect, the present invention relates to a radionuclidecomplex of a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein L is a leavinggroup. L may be OR. R may be selected from C₁ to C₁₀ alkyl, C₁ to C₁₀heteroalkyl, C₂ to C₁₀ alkene, C₂ to C₁₀ alkyne, and aryl, each of whichis optionally substituted. R may be alkyl (e.g. C₁ to C₆ alkyl, such asmethyl, ethyl, propyl or butyl). R may be methyl or ethyl. R may beethyl.

The radionuclide may be a radioisotope of zirconium, gallium or indium.The radioisotope of zirconium may be ⁸⁹Zr. The radioisotope of galliummay be ⁶⁸Ga. The radioisotope of indium may be ¹¹¹In. The radionuclidemay be a radioisotope of zirconium (e.g. ⁸⁹Zr).

In another aspect, the present invention also relates to a conjugate of:

-   -   a compound of formula (I):

or a pharmaceutically-acceptable salt thereof, wherein L is a leavinggroup (as defined herein), and

-   -   a target molecule.

The target molecule may be a polypeptide (such as a transfer protein oran antibody). The target molecule may be a peptide (such as a targetingpeptide). The polypeptide may be an antibody. The antibody may beselected from Herceptin (trastuzumab), rituximab and cetuximab.

In another aspect, the present invention relates to aradionuclide-labelled conjugate of:

-   -   a compound of formula (I):

or a pharmaceutically-acceptable salt thereof, wherein L is a leavinggroup (as defined herein),

-   -   a target molecule, and    -   a radionuclide complexed thereto.

The target molecule may be a polypeptide (such as a transfer protein oran antibody). The target molecule may be a peptide (such as a targetingpeptide). The polypeptide may be an antibody. The antibody may beselected from Herceptin (trastuzumab), rituximab and cetuximab.

The radionuclide may be a radioisotope of zirconium, gallium or indium.The radioisotope of zirconium may be ⁸⁹Zr. The radioisotope of galliummay be ⁶⁸Ga. The radioisotope of indium may be ¹¹¹In. The radionuclidemay be an isotope of zirconium (e.g. ⁸⁹Zr).

The radionuclide-labelled complex and the radionuclide-labelledconjugate have improved affinity when compared to with DFO-Ph-NCS or aconjugate of DFO-Ph-NCS and the target molecule.

In another aspect, the present invention relates to a method of imaginga patient, the method including:

-   -   administering to a patient a radionuclide-labelled conjugate, as        defined above, and    -   imaging the patient.

In another aspect, the present invention relates to a method of imaginga cell or in vitro biopsy sample, the method including:

-   -   administering to a cell or in vitro biopsy sample a        radionuclide-labelled conjugate, as defined above, and    -   imaging the cell or in vitro biopsy sample.

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Micro-PET imaging of HER2-positive tumour (BT474 breastcarcinoma model) using ⁸⁹Zr(DFO-squarate-trastuzumab).

FIG. 2. Micro-PET imaging of HER2-positive tumour (LS174T colorectaltumour model) using ⁸⁹Zr(DFO-maleimide-trastuzumab).

FIG. 3. Micro-PET imaging of HER2-positive tumour (LS174T colorectaltumour model) using ⁸⁹ZrCl.

FIG. 4. A radio-iTLC chromatogram of a control sample (i.e. no DFOSq).

FIG. 5. A radio-iTLC chromatogram of the ⁸⁹Zr DFOSq complex (60 minutesafter addition of ⁸⁹Zr).

FIGS. 6A-6C. Two Size Exclusion HPLC UV-Vis chromatograms (at twodifferent absorption wavelengths of 280 (FIG. 6A) and 254 nm (FIG. 6B))and a radiation chromatogram of a control sample (FIG. 6C).

FIGS. 7A-7C. Two Size Exclusion HPLC UV-Vis chromatograms (at twodifferent absorption wavelengths of 280 (FIG. 7A) and 254 nm (FIG. 7B))and a radiation chromatogram (FIG. 7C) of the ⁸⁹Zr DFOSq complex (78hours after addition of ⁸⁹Zr).

FIG. 8. A radio-iTLC chromatogram of ⁸⁹Zr-labelled DFOSq-cRGDfK (taken60 minutes after addition of ⁸⁹Zr).

FIG. 9. An LCMS spectrum of DFOSq-transferrin (DFOSq-Tf).

FIG. 10. A radio-iTLC chromatogram of ⁸⁹Zr-labelled DFOSq-Tf (taken 20minutes after addition of ⁸⁹Zr).

FIGS. 11A-110. Two Size Exclusion HPLC UV-Vis chromatograms (at twodifferent absorption wavelengths of 280 (FIG. 11A) and 254 nm (FIG.11B)) and a radiation chromatogram (FIG. 11C) of ⁸⁹Zr-labelled DFOSq-Tf(20 minutes after addition of ⁸⁹Zr).

FIG. 12. An LCMS spectrum of DFOSq-herceptin (DFOSq-Herc).

FIG. 13. A radio-iTLC chromatogram of ⁸⁹Zr-labelled DFOSq-Herc (taken 25minutes after addition of ⁸⁹Zr).

FIG. 14. A radio-iTLC chromatogram of purified ⁸⁹Zr-labelled DFOSq-Herc.

FIGS. 15A-150. Two Size Exclusion HPLC UV-Vis chromatograms (at twodifferent absorption wavelengths of 280 (FIG. 15A) and 254 nm (FIG.15B)) and a radiation chromatogram (FIG. 15C) of cold (i.e. unlabelled)DFOSq-Herc.

FIGS. 16A-160. Two Size Exclusion HPLC UV-Vis chromatograms (at twodifferent absorption wavelengths of 280 (FIG. 16A) and 254 nm (FIG.16B)) and a radiation chromatogram (FIG. 16C) of ⁸⁹Zr-labelledDFOSq-Herc (taken 24 hours after purification).

FIG. 17. PET image of mouse 1 after administration of⁸⁹ZrDFOSq-Herceptin.

FIG. 18. PET image of mouse 2 after administration of⁸⁹ZrDFOSq-Herceptin.

FIG. 19. Deconvoluted ESI-MS of DFOSq-trastuzumab (unlabelledtrastuzumab=148,232).

FIG. 20. iTLC analysis of ⁸⁹Zr-DFOPhNCS-trastuzumab reaction mixtureafter 30 min (origin is at 55 mm, solvent front at 150 mm; labelledtrastuzumab remains at the origin, activity with a distance of >70 mm(r.f. >0.1) represents non-chelated ⁸⁹Zr).

FIGS. 21A-21B. SE-HPLC analysis of ⁸⁹Zr-DFOSq-trastuzumab after PD-10purification (FIG. 21A: absorbance at 280 nm; FIG. 21B: radiation signal(my); ⁸⁹Zr-DFOSq-trastuzumab elutes at ˜12 mins, gentisate elutes at20-25 mins).

FIG. 22. PET imaging of BT474 tumour bearing NOD/SCID mice using⁸⁹Zr-DFOSq-trastuzumab.

FIG. 23. Deconvoluted ESI-MS of DFOSq-trastuzumab (unlabelledtrastuzumab=148,232).

FIG. 24. iTLC analysis of ⁸⁹Zr-DFOSq-trastuzumab reaction mixture after1 hr (origin is at 70 mm, solvent front at 160 mm; labelled trastuzumabremains at the origin, activity with a distance of >80 mm (r.f. >0.1)represents non-chelated ⁸⁹Zr).

FIG. 25. Radiation trace by SE-HPLC analysis of the purified⁸⁹Zr-DFOSq-trastuzumab (product retention time ˜12.5 min).

FIG. 26. iTLC analysis of ⁸⁹Zr-DFOSq-trastuzumab reaction mixture after1.5 hr (origin is at 70 mm, solvent front at 145 mm; labelledtrastuzumab remains at the origin, activity with a distance of >80 mm(r.f. >0.1) represents non-chelated ⁸⁹Zr).

FIG. 27. Radiation trace by SE-HPLC analysis of the purified⁸⁹Zr-DFOSq-trastuzumab (product retention time ˜12.5 min).

FIG. 28. PET imaging of SKOV3 tumour-bearing mice using⁸⁹Zr-DFOSq-trastuzumab as the imaging agent.

FIG. 29. PET imaging of LS174T tumour-bearing mice using⁸⁹Zr-DFOSq-trastuzumab as the imaging agent.

FIG. 30. ¹H NMR analysis of DFOPhNCS (d₆-DMSO, 400 MHz).

FIG. 31. Analytical HPLC trace (absorbance at 214 nm) of purifiedDFOPhNCS (signal at 1.5 min=DMSO, 8.95 min=DFOPhNCS).

FIG. 32. ESI-MS analysis of purified DFOPhNCS.

FIG. 33. Deconvoluted ESI-MS of DFOPhNCS-trastuzumab (unlabelledtrastuzumab=148,234; trastuzumab with one DFOPhNCS attachment=148,987).

FIGS. 34A-34C. FIG. 34A shows iTLC analysis of ⁸⁹Zr-DFOPhNCS-trastuzumabreaction mixture after 1 hr, showing ˜30% labelling efficiency (originis at 60 mm, solvent front at 150 mm; labelled trastuzumab remains atthe origin, activity with a distance of >70 mm (r.f. >0.1) representsnon-chelated ⁸⁹Zr). FIG. 34B shows iTLC analysis of⁸⁹Zr-DFOPhNCS-trastuzumab reaction mixture after 1.5 hr, showing ˜50%labelling efficiency (origin is at 55 mm, solvent front at 135 mm;Labelled trastuzumab remains at the origin, activity with a distanceof >65 mm (r.f. >0.1) represents non-chelated ⁸⁹Zr). FIG. 34C shows iTLCanalysis of ⁸⁹Zr-DFOPhNCS-trastuzumab reaction mixture after 2 hr,showing ˜65% labelling efficiency (origin is at 60 mm, solvent front at145 mm; labelled trastuzumab remains at the origin, activity with adistance of >70 mm (r.f. >0.1) represents non-chelated ⁸⁹Zr).

FIG. 35. iTLC analysis of ⁸⁹Zr-DFOPhNCS-trastuzumab after PD-10purification (origin is at 55 mm, solvent front at 150 mm; labelledtrastuzumab remains at the origin, activity with a distance of >70 mm(r.f. >0.1) represents non-chelated ⁸⁹Zr).

FIGS. 36A-36B. SEC-HPLC analysis of ⁸⁹Zr-DFOPhNCS-trastuzumab afterPD-10 purification (FIG. 36A: absorbance at 280 nm; FIG. 36B: radiationsignal (my); ⁸⁹Zr-DFOPhNCS-trastuzumab begins to elute at ˜12 mins,gentisic acid elutes at 20-25 mins).

FIG. 37. PET imaging of SKOV3 tumour bearing mice using⁸⁹Zr-DFOPhNCS-trastuzumab as the imaging agent.

FIG. 38. PET imaging results of ⁸⁹Zr-DFO-Sq/Herceptin vs⁸⁹Zr-DFO-Ph-NCS/Herceptin uptake in SKOV3 tumour-bearing mice.

FIGS. 39A-39B. HPLC analysis of ⁸⁹Zr-DFOPhNCS-cRGDfK reaction mixture;FIG. 39A: absorbance at 280/254 nm; FIG. 39B: radiation.

FIGS. 40A-40C. HPLC analysis of ⁸⁹Zr-DFOSq-cRGDfK reaction mixture; FIG.40A: absorbance at 254; FIG. 40B: absorbance at 280 nm; FIG. 40C:radiation.

FIGS. 41A-41B. HPLC analysis of DFOPhNCS-cRGDfK/DFOSq-cRGDfK/⁸⁹Zrreaction mixture; FIG. 41A: absorbance at 280/254 nm; FIG. 41B:radiation.

FIG. 42. ¹H NMR (400 MHz, D₂O) spectrum of DFOSqTaur.

FIG. 43. ESI-MS spectrum of DFOSqTaur. [M+H]+ (calc) m/z=764.35.

FIG. 44. ¹H NMR (400 MHz, D₂O) spectrum of DFOPhSO₃H.

FIG. 45. ESI-MS spectrum of DFOPhSO3H. [M+H]+ (calc) m/z=776.33.

FIG. 46. ESI-MS spectrum of DFOPhSO₃H/DFOSqTaur/Zr reaction mixture.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present inventors have found that the synthesis of the compound offormula (I) (and, in particular, the synthesis of conjugates of thiscompound with biological molecules) is simpler and more amendable to thepresence of pH-sensitive molecules, such as antibodies, than that ofN-suc-DFO-TFP, as it is not necessary to use Fe³⁺ ions to protect thehydroxamic acid groups. This makes antibody conjugation andradiolabelling with ⁸⁹Zr straightforward. In addition, the compound offormula (I) is not sensitive to chloride-containing buffers, does notresult in the formation of aggregates during conjugation withbiomolecules, and does not bind reversibly to biological molecules.

In addition, unexpectedly, the radiolabelled conjugates of the compoundsof formula (I) with target molecules exhibit improved tumour targetingand tissue selectivity over a number of the known radionuclide chelators(particularly other DFO-based chelators) that are used as PET imagingagents.

There are a number of potential factors that could contribute to thisimproved tumour targeting and tissue selectivity, including the strengthwith which the radioisotope is chelated, metabolic stability, and theexcretion rate of metabolites. None of these advantages of the compoundsof the present invention are disclosed in the prior art, nor could theyexpected, and the relative weighting of their contributions to theoverall improved performance of the compounds of the present inventionis unknown.

A “pharmaceutically acceptable salt” of a compound disclosed herein isan acid or base salt that is generally considered in the art to besuitable for use in contact with the tissues of human beings or animalswithout excessive toxicity or carcinogenicity, and preferably withoutirritation, allergic response, or other problem or complication. Suchsalts include mineral and organic acid salts of basic residues such asamines, as well as alkali or organic salts of acidic residues such ascarboxylic acids.

Suitable pharmaceutically acceptable salts include, but are not limitedto, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic,glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic,toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic,2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic,succinic, fumaric, maleic, propionic, hydroxymaieic, hydroiodic,phenylacetic, alkanoic (such as acetic, HOOC—(CH₂)_(n)—COOH where n isany integer from 0 to 6, i.e. 0, 1, 2, 3, 4, 5 or 6), and the like.Similarly, pharmaceutically acceptable cations include, but are notlimited to sodium, potassium, calcium, aluminum, lithium and ammonium. Aperson skilled in the art will recognize further pharmaceuticallyacceptable salts for the compounds provided herein. In general, apharmaceutically acceptable acid or base salt can be synthesized from aparent compound that contains a basic or acidic moiety by anyconventional chemical method. Briefly, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent (such as ether, ethyl acetate, ethanol, isopropanol oracetonitrile), or in a mixture of the two.

It will be apparent that the compounds of formula (I) may, but need not,be present as a hydrate, solvate or non-covalent complex (to a metalother than the radionuclide). In addition, the various crystal forms andpolymorphs are within the scope of the present invention, as areprodrugs of the compounds provided herein.

A “prodrug” is a compound that may not fully satisfy the structuralrequirements of the compounds provided herein, but is modified in vivo,following administration to a subject or patient, to produce aradiolabelled conjugate as provided herein. For example, a prodrug maybe an acylated derivative of a radiolabelled conjugate. Prodrugs includecompounds wherein hydroxyl or amine groups are bonded to any group that,when administered to a mammalian subject, cleaves to form a freehydroxyl or amine group, respectively. Examples of prodrugs include, butare not limited to, acetate, formate, phosphate and benzoate derivativesof amine functional groups within the radiolabelled conjugate. Prodrugsof the may be prepared by modifying functional groups present in thecompounds in such a way that the modifications are cleaved in vivo togenerate the parent compounds.

A “substituent” as used herein, refers to a molecular moiety that iscovalently bonded to an atom within a molecule of interest. The term“substituted,” as used herein, means that any one or more hydrogens onthe designated atom is replaced with a selection from the indicatedsubstituents, provided that the designated atom's normal valence is notexceeded, and that the substitution results in a stable compound, i.e.,a compound that can be isolated, characterized and tested for biologicalactivity. When a substituent is oxo, i.e., ═O, then two hydrogens on theatom are replaced. An oxo group that is a substituent of an aromaticcarbon atom results in a conversion of —CH— to —C(═O)— and a loss ofaromaticity. For example a pyridyl group substituted by oxo is apyridone. Examples of suitable substituents are alkyl, heteroalkyl,halogen (for example, fluorine, chlorine, bromine or iodine atoms), OH,═O, SH, SO₂, NH₂, NHalkyl, ═NH, N₃ and NO₂ groups.

The term “optionally substituted” refers to a group in which one, two,three or more hydrogen atoms have been replaced independently of eachother by alkyl, halogen (for example, fluorine, chlorine, bromine oriodine atoms), OH, ═O, SH, ═S, SO₂, NH₂, NHalkyl, ═NH, N₃ or NO₂ groups.

As used herein a wording defining the limits of a range of length suchas, for example, “from 1 to 5” means any integer from 1 to 5, i. e. 1,2, 3, 4 and 5. In other words, any range defined by two integersexplicitly mentioned is meant to comprise and disclose any integerdefining said limits and any integer comprised in said range.

The term “leaving group” refers to any moiety that is capable of beingdisplaced from the squarate moiety upon reaction with a target molecule.The leaving group will be displaced and a bond will form between a group(such as an amino group of a lysine side chain) of the target moleculeand the squarate. In one embodiment, the leaving group (“L”) is OR. Inone embodiment, R is selected from C₁ to C₁₀ alkyl, C₁ to C₁₀heteroalkyl, C₂ to C₁₀ alkene and C₂ to C₁₀ alkyne, and aryl, each ofwhich is optionally substituted. In one embodiment, R is C₁ to C₁₀ alkyl(e.g. C₁ to C₆ alkyl, such as methyl, ethyl, propyl or butyl). In oneembodiment, R is methyl or ethyl. In one embodiment, R is ethyl. Inanother embodiment, L is halogen (e.g. fluorine, chlorine, bromine oriodine), or L is an azide group.

The term “alkyl” refers to a saturated, straight-chain or branchedhydrocarbon group that contains from 1 to 10 carbon atoms, for example an-octyl group, especially from 1 to 6, i.e. 1, 2, 3, 4, 5, or 6, carbonatoms. Specific examples of alkyl groups are methyl, ethyl, propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,iso-pentyl, n-hexyl and 2,2-dimethylbutyl.

The term “heteroalkyl” refers to an alkyl group as defined above thatcontains one or more heteroatoms selected from oxygen, nitrogen andsulphur. Specific examples of heteroalkyl groups are methoxy,trifluoromethoxy, ethoxy, n-propyloxy, iso-propyloxy, butoxy,tert-butyloxy, methoxymethyl, ethoxymethyl, —CH₂CH₂OH, —CH₂OH,methoxyethyl, 1-methoxyethyl, 1-ethoxyethyl, 2-methoxyethyl or2-ethoxyethyl, methylamino, ethylamino, propylamino, iso-propylamino,dimethylamino, diethylamino, iso-propyl-ethylamino, methylamino methyl,ethylamino methyl, di-iso-propylamino ethyl, methylthio, ethylthio,iso-propylthio, methanesulfonyl, trifluoromethanesulfonyl, enol ether,dimethylamino methyl, dimethylamino ethyl, acetyl, propionyl,butyryloxy, acetyloxy, methoxycarbonyl, ethoxy-carbonyl, propionyloxy,acetylamino, propionylamino, carboxymethyl, carboxyethyl orcarboxypropyl, N-ethyl-N-methylcarbamoyl and N-methylcarbamoyl. Furtherexamples of heteroalkyl groups are nitrile, iso-nitrile, cyanate,thiocyanate, iso-cyanate, iso-thiocyanate and alkylnitrile groups.

The term “alkenyl” refers to an at least partially unsaturated,straight-chain or branched hydrocarbon group that contains from 2 to 10carbon atoms, especially from 2 to 6, i.e. 2, 3, 4, 5 or 6, carbonatoms. Specific examples of alkenyl groups are ethenyl (vinyl), propenyl(allyl), iso-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl,propargyl, iso-prenyl and hex-2-enyl group. Preferably, alkenyl groupshave one or two double bond(s).

The term “alkynyl” refers to an at least partially unsaturated,straight-chain or branched hydrocarbon group that contains from 2 to 10carbon atoms, especially from 2 to 6, i.e. 2, 3, 4, 5 or 6, carbonatoms. Specific examples of alkynyl groups are ethynyl, propynyl,butynyl, acetylenyl and propargyl groups. Preferably, alkynyl groupshave one or two (especially preferably one) triple bond(s).

The term “aryl” refers to an aromatic group that contains one or morerings containing from 6 to 14 ring carbon atoms, preferably from 6 to 10(especially 6) ring carbon atoms. Examples are phenyl, naphthyl andbiphenyl groups. Examples of substituted aryl groups suitable for use inthe present invention include p-toluenesulfonyl (Ts), benzenesulfonyl(Bs) and m-nitrobenzenesulfonyl (Ns).

Preferred compounds of the present invention are those where R is C₁ toC₁₀ alkyl (and in particular, where R is ethyl).

In one embodiment, the leaving group is selected from OCH₂CH₃,O-p-toluenesulfonate (OTs), O-methanesulfonate (OMs),O-trifluoromethanesulfonate (OTf), O-benzenesulfonate (OBs),O-m-nitrobenzenesulfonate (ONs), cyanate (CN), azide (N₃) and halogen(e.g. fluorine, chlorine, bromine or iodine).

As used herein, the term “radionuclide complex” refers to a compound offormula (I), as defined above, which has formed a co-ordination complexwith a radionuclide. Generally, this occurs as a result of the formationof co-ordination bonds between the electron donating groups (such as thehydroxamate groups) of the compound of formula (I) and the radionuclide.

In the compounds of the present invention, co-ordination bonds arepostulated to form between the hydroxamic acid groups of the DFO and theradionuclide. However, without wishing to be bound by theory, thepresent inventors also believe that the oxo groups on the squaratemoiety (in addition to the hydroxamic acid groups of DFO) also act asdonor atoms, providing one or two additional sites by which the compoundof formula (I) can bind to the radionuclide. This results in aneight-coordinate complex, which is very favourable from a stabilityperspective for radionuclides that have eight-coordinate geometry (suchas ⁸⁹Zr), and may explain the stability observed in respect of thecomplexes of the present invention. In particular, it may explain whythe radionuclide does not as readily leach out of the target tissue(into other tissue, such as bone) therefore resulting in improvedimaging quality when compared to other DFO-based imaging agents. Theseadvantages of the compounds of the present invention over thecurrently-used chelators are illustrated in the Figures and Examples.

As shown in FIGS. 1, 18 and 22, ⁸⁹Zr(DFOSq-trastuzumab) very selectivelytargets, and remains concentrated at the site of, the HER2-positivetumour BT474 (a breast carcinoma). This is in contrast to the resultsshown in FIGS. 2 and 3, which demonstrate significant distribution ofthe radionuclide (when administered as ⁸⁹Zr(DFO-maleimide-trastuzumab)and ⁸⁹ZrCl, respectively) throughout the bodies of the mice. Asdiscussed above, one possible contributor to the improved specificity isthat the squarate-based agent has strong chelating potential therebypreventing its distribution throughout the body and accumulation inother tissue, such as bone (for which zirconium has a very highaffinity), the liver and the kidneys.

The high affinity for zirconium as compared with ⁸⁹Zr(DFO-PhNCS) isdemonstrated in a competition study (see the Examples and FIGS. 39 to41). The absorbance and radiation spectra show that ⁸⁹Zr is complexedalmost exclusively by the conjugate of the present invention when ⁸⁹Zris exposed to a mixture of a conjugate of the present invention(DFOSq-cRGDfK) with DFOPhNCS-cRGDfK.

Another contributor to the specificity may be the metabolic stability ofthe radionuclide-labelled compound of the present invention. Metabolitesthat contain the radionuclide but that are free of the trastuzumabmoiety (“non-targeted metabolites”) would have no targeting ability,resulting in distribution of the radionuclide throughout the body. Thismetabolic stability of the compounds of the present invention could notbe expected, and cannot readily be explained.

The present inventors also postulate that, even if metabolites of theradionuclide-labelled conjugates of the present invention are formed,they may have a high excretion rate, leading to less accumulation of theradionuclide at non-target sites. This would also be an unexpectedproperty.

This specificity and stability of the conjugate of the present inventionis also illustrated in FIGS. 28 and 29, which demonstrate the imagingability of ⁸⁹Zr(DFOSq-trastuzumab) in respect of other HER2-positivetumours (LS174T, which is a colorectal tumour model, and SKOV3, which isan ovarian cancer model). The results obtained with⁸⁹Zr(DFOSq-trastuzumab) in the SKOV3 tumour model (FIG. 28) can also bequalitatively contrasted with those obtained in the same tumour modelbut using ⁸⁹Zr(DFO-PhNCS-trastuzumab) as the imaging agent (see FIG.37), which shows significant distribution of the radionuclide throughoutthe treated mice.

The superior activity of the radiolabelled conjugate of the presentinvention over other conjugates, such as ⁸⁹Zr(DFO-PhNCS-trastuzumab) isalso demonstrated by the Standardized Uptake Values (SUVs) obtained fromthe imaging of SKOV3 tumours using ⁸⁹Zr(DFO-squarate-trastuzumab) (seeTable 3 in the Examples) compared with the SUVs obtained from theimaging of SKOV3 tumours using ⁸⁹Zr(DFO-PhNCS-trastuzumab) (see Table 7in the Examples). SUV is basically the tissue radioactivityconcentration (at time point t), divided by the injected activitydivided by the body weight of the animal. Therefore, the SUVstandardises for different amounts of radioactivity injected and thesize of the animal.

In general, the best images (and less radio toxicity to non-targetorgans) are obtained when the ratio of radioimaging agent uptake in atumour to the uptake of the agent by non-target tissue (such as bone andthe liver) is higher. The higher the ratio, the better the image andselectivity of the radioimaging agent. The graphs in FIG. 38 show thetumour SUV_(max), as well as the SUV ratio for tumour:background,tumour:liver and tumour:bone, for a radiolabelled conjugate of thepresent invention (⁸⁹Zr(DFOSq-trastuzumab)), and⁸⁹Zr(DFO-PhNCS-trastuzumab). From FIG. 38, it can be seen that the SUVratio of ⁸⁹Zr(DFOSq-trastuzumab is higher across all experiments thanthe SUV ratio of ⁸⁹Zr(DFO-PhNCS-trastuzumab) because there is moreradioactivity at the target site than in the other tissue (liver andbone). This demonstrates that the radiolabelled conjugate of the presentinvention is a more selective and stable agent than⁸⁹Zr(DFO-PhNCS-trastuzumab). The high tumour:background ratio isadvantageous.

Notably, the uptake of the radiolabelled conjugate of the presentinvention is also dependent on the HER2 expression level of the tumours.The data presented here demonstrate that tumours that have a high HER2expression level (such as BT474) have greater uptake of the conjugate(and therefore produce a stronger PET image) than tumours that have alower level of HER2 expression (e.g. LS174T, which will result in a“dimmer” image). This difference in image strength as a result ofvarying the HER2 expression level is a strong indication that it is theHER2 expression level on a tumour that influences the strength of thePET image obtained, not the presence of different metabolites,

As used herein, the term “radionuclide” (also commonly referred to as aradioisotope or radioactive isotope), is an atom with an unstablenucleus. It radioactively decays resulting in the emission of nuclearradiation (such as gamma rays and/or subatomic particles such as alphaor beta particles). In one embodiment, the radionuclide is one that isalso useful in radioimmunotherapy applications (e.g. a beta particleemitter). Preferably, the radionuclide has eight-coordinate geometry.Examples of radionuclides suitable for use in the present inventioninclude radioisotopes of zirconium (e.g. ⁸⁹Zr), gallium (e.g. ⁶⁷Ga and⁶⁸Ga), lutetium (e.g. ¹⁷⁶Lu and ¹⁷⁷Lu), holmium (e.g. ¹⁶⁶Ho), scandium(e.g. ⁴⁴Sc and ⁴⁷Sc), titanium (e.g. ⁴⁵Ti), indium (e.g. ¹¹¹1 n and¹¹⁵In), yttrium (e.g. ⁸⁶Y and ⁹⁰Y), terbium e.g. (¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tband ¹⁶¹Tb), technetium (e.g. ^(99m)Tc), samarium (e.g. ¹⁵³Sm) andniobium (e.g. ⁹⁵Nb and ⁹⁰Nb). The radionuclide for use in the presentinvention may be selected from gallium (specifically, ⁶⁷Ga and ⁶⁸Ga),indium (specifically, ¹¹¹In), and zirconium (specifically, ⁸⁹Zr). Theradionuclide for use in the present invention may be selected from ⁶⁸Ga,¹¹¹In and ⁸⁹Zr. For example, ⁶⁸Ga has been shown to bind with DFO (seeUeda et al (2015) Mol Imaging Biol, vol. 17, pages 102-110), and indiumhas similar co-ordination chemistry to zirconium (and therefore would beexpected to bind to the compound of formula (I) in a similar way).

It will be understood by a person skilled in the art that the compoundof the present invention can also complex non-radioactive metals used inimaging applications, such as MRI. An example of such a metal isgadolinium (e.g. ¹⁵²Gd).

As mentioned above, the present invention also relates to a conjugate ofa compound of formula (I), or a pharmaceutically-acceptable saltthereof, and a target molecule.

As used herein, the term “target molecule” refers to a biologicalmolecule, or a fragment of a biological molecule, that has the abilityto target a particular tissue or tumour. The target molecule may be apolypeptide, such as a protein (e.g. a transport protein such astransferrin), an albumin (e.g. serum albumin) or an antibody (e.g.trastuzumab, also known as herceptin, ranibizumab, bevacizumab,fresolimumab, cetuximab, panitumumab, rituximab, pertuzumab, andofatumumab). The antibody may be selected from Herceptin, rituximab andcetuximab. The target molecule may be a peptide (e.g. a targetingpeptide that is used to target cells involved in tumour angiogenesis,such as cyclic RGD sequences, or another targeting peptide, such asoctreotate, bombesin and glu-N(CO)N-lys PSMA). The target molecule willhave a functional group (such as an amine group of a lysine residue)that will react with the squarate moiety to form a covalent link betweenthe target molecule and the compound of formula (I). This results information of the conjugate. The conjugate may also include aradionuclide complexed thereto. This produces a radionuclide-labelledconjugate of a compound of formula (I), or a pharmaceutically-acceptablesalt thereof, a target molecule, and a radionuclide complexed thereto.In one embodiment, the radionuclide is a radioisotope of zirconium (e.g.⁸⁹Zr).

The compounds of formula (I) and the radionuclide complexes can besynthesised by any suitable method known to a person skilled in the art.An example of a synthetic method is given below in Scheme 1.

The radionuclide complexes can be conjugated with the target moleculesof interest (to produce a radiolabelled conjugate) by any suitablemethod known to a person skilled in the art. An example of a suitablemethod is set out as follows:

-   -   1. Prepare target molecule in borate buffer, pH 9, at a        concentration such that the final buffer concentration of the        reaction mixture is 0.5 M.    -   2. Prepare DFOSq solution in 4% DMSO in MilliQ water (DMSO        should be added first to ensure the DFOSq is fully dissolved).    -   3. DFOSq solution should be added to the target molecule as        required and the reaction mixture left to stand at room        temperature overnight (shorter reaction times will result in        lower average chelators per target molecule).    -   4. Conjugates can be purified using spin filters with an        appropriate molecular weight limit (at least 1 kDa). After        initial filtration the conjugate should be washed on the spin        filter at least twice with 4% DMSO in MilliQ to remove all        excess DFOSq.    -   5. A buffer exchange step using the spin filter then allows for        storage of the conjugate (0.9% NaCl solution is recommended for        DFOSq-Herceptin).

It will also be clear to a person skilled in the art that the conjugatecan be prepared in the absence of the radionuclide. In this embodiment,the radionuclide is added to the conjugate once the conjugate has beenprepared.

The present invention also relates to pharmaceutical compositionsincluding a radionuclide-labelled conjugate of:

-   -   a compound of formula (I):

or a pharmaceutically-acceptable salt thereof, wherein L is a leavinggroup (as defined herein),

-   -   a target molecule, and    -   a radionuclide complexed thereto,

and one or more pharmaceutically acceptable carrier substances,excipients and/or adjuvants.

Pharmaceutical compositions may include, for example, one or more ofwater, buffers (for example, neutral buffered saline, phosphate bufferedsaline, citrates and acetates), ethanol, oil, carbohydrates (forexample, glucose, fructose, mannose, sucrose and mannitol), proteins,polypeptides or amino acids such as glycine, antioxidants (e.g. sodiumbisulfite), tonicity adjusting agents (such as potassium and calciumchloride), chelating agents such as EDTA or glutathione, vitamins and/orpreservatives.

Pharmaceutical compositions will preferably be formulated for parenteraladministration. The term “parenteral” as used herein includessubcutaneous, intradermal, intravascular (for example, intravenous),intramuscular, spinal, intracranial, intrathecal, intraocular,periocular, intraorbital, intrasynovial and intraperitoneal injection,as well as any similar injection or infusion technique. Intravenousadministration is preferred. Suitable components of parenteralformulations, and methods of making such formulations, are detailed invarious texts, including “Remington's Pharmaceutical Sciences”.

The composition of the present invention will be administered to apatient parenterally in the usual manner. The DFO-squaramide conjugatecomplex may then take anywhere from 1 hour to 24 hours to distributethroughout the body to the target site. Once the desired distributionhas been achieved, the patient will be imaged.

Accordingly, the present invention also relates to a method of imaging apatient, the method including:

-   -   administering to a patient the radionuclide-labelled conjugate,        as defined herein; and    -   imaging said patient.

The present invention also relates to a method of imaging a cell or invitro biopsy sample, the method including:

-   -   administering to a cell or in vitro biopsy sample the        radionuclide-labelled conjugate, as defined herein; and    -   imaging the cell or in vitro biopsy sample.

Preferably, the target molecule serves to target the conjugate to adesired site in vivo, or to a desired site in the cell or in the biopsysample. Preferably, the desired site is a tumour.

It will be understood, that the specific dose level for any particularpatient, and the length of time that the agent will take to arrive atthe target site, will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, and the severity of theparticular disorder undergoing therapy.

The term “effective amount” refers to an amount of the that results in adetectable amount of radiation following administration of theradionuclide-labelled conjugate to a patient. A person skilled in theart will know how much of the radionuclide-labelled conjugate toadminister to a patient to achieve the optimal imaging capabilitywithout causing problems from a toxicity perspective. Theradionuclide-labelled conjugates of the present invention findparticular use in assisting clinicians to determine where a cancer is(including whether a target, such a receptor, is homogeneously presenton a tumour), what treatment a cancer will respond to (which facilitatestreatment selection and determination of optimal dosages), and how muchof the treatment will ultimately reach the target site. Theradionuclide-labelled conjugates of the present invention can also beused to study the pharmacokinetics and biodistribution of particulartarget molecules (e.g. during drug development of new biologicaltherapeutic agents, such as monoclonal antibodies).

Patients may include but are not limited to primates, especially humans,domesticated companion animals such as dogs, cats, horses, and livestocksuch as cattle, pigs and sheep, with dosages as described herein.

As mentioned above, the radionuclide-labelled conjugates of the presentinvention are particularly useful for imaging tumours (which form as aresult of uncontrolled or progressive proliferation of cells). Some suchuncontrolled proliferating cells are benign, but others are termed“malignant” and may lead to death of the organism. Malignant neoplasmsor “cancers” are distinguished from benign growths in that, in additionto exhibiting aggressive cellular proliferation, they may invadesurrounding tissues and metastasize. Moreover, malignant neoplasms arecharacterized in that they show a greater loss of differentiation(greater “dedifferentiation”), and greater loss of their organizationrelative to one another and their surrounding tissues. This property isalso called “anaplasia”. Neoplasms treatable by the present inventionalso include solid phase tumors/malignancies, i.e. carcinomas, locallyadvanced tumors and human soft tissue sarcomas. Carcinomas include thosemalignant neoplasms derived from epithelial cells that infiltrate(invade) the surrounding tissues and give rise to metastatic cancers,including lymphatic metastases.

Adenocarcinomas are carcinomas derived from glandular tissue, or whichform recognizable glandular structures. Another broad category ofcancers includes sarcomas, which are tumors whose cells are embedded ina fibrillar or homogeneous substance like embryonic connective tissue.

The type of cancer or tumor cells that may be amenable to imagingaccording to the invention include, for example, breast, colon, lung,and prostate cancers, gastrointestinal cancers including esophagealcancer, stomach cancer, colorectal cancer, polyps associated withcolorectal neoplasms, pancreatic cancer and gallbladder cancer, cancerof the adrenal cortex, ACTH-producing tumor, bladder cancer, braincancer including intrinsic brain tumors, neuroblastomas, astrocyticbrain tumors, gliomas, and metastatic tumor cell invasion of the centralnervous system, Ewing's sarcoma, head and neck cancer including mouthcancer and larynx cancer, kidney cancer including renal cell carcinoma,liver cancer, lung cancer including small and non-small cell lungcancers, malignant peritoneal effusion, malignant pleural effusion, skincancers including malignant melanoma, tumor progression of human skinkeratinocytes, squamous cell carcinoma, basal cell carcinoma, andhemangiopericytoma, mesothelioma, Kaposi's sarcoma, bone cancerincluding osteomas and sarcomas such as fibrosarcoma and osteosarcoma,cancers of the female reproductive tract including uterine cancer,endometrial cancer, ovarian cancer, ovarian (germ cell) cancer and solidtumors in the ovarian follicle, vaginal cancer, cancer of the vulva, andcervical cancer, breast cancer (small cell and ductal), penile cancer,retinoblastoma, testicular cancer, thyroid cancer, trophoblasticneoplasms, and Wilms' tumor.

It may also be advantageous to administer the radionuclide-labelledconjugates of the present invention with drugs that have anti-canceractivity. Examples of suitable drugs in this regard includefluorouracil, imiquimod, anastrozole, axitinib, belinostat, bexarotene,bicalutamide, bortezomib, busulfan, cabazitaxel, capecitabine,carmustine, cisplatin, dabrafenib, daunorubicin hydrochloride,docetaxel, doxorubicin, eloxati, erlotinib, etoposide, exemestane,fulvestrant, methotrexate, gefitinib, gemcitabine, ifosfamide,irinotecan, ixabepilone, lanalidomide, letrozole, lomustine, megestrolacetate, temozolomide, vinorelbine, nilotinib, tamoxifen, oxaliplatin,paclitaxel, raloxifene, pemetrexed, sorafenib, thalidomide, topotecan,vermurafenib and vincristine.

The radionuclide-labelled conjugates of the present invention can alsobe used to determine whether a particular tumour has one or more typesof receptor, and therefore whether a patient may benefit from aparticular therapy. For example, by using herceptin as a target moleculein the radiolabelled conjugate, the presence of HER2 receptors on apatient's tumour can be tested for. If the tumour is HER2-negative (i.e.does not have HER2 receptors), the imaging agent will not “stick” to thetumour, indicating that herceptin may not be a useful therapy for thepatient.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

EXAMPLES

All reagents and solvents were obtained from standard commercial sourcesand unless otherwise stated were used as received.

¹H and ¹³C spectra were recorded with a Varian FT-NMR 400 or VarianFT-NMR 500 (Varian, California USA). ¹H-NMR spectra were acquired at 400or 500 MHz and ¹³C-NMR spectra were acquired at 101 or 125 MHz. All NMRspectra were recorded at 25° C. unless otherwise stated. The reportedchemical shifts (in parts per million) are referenced relative toresidual solvent signal.

ESI-MS of non-protein samples were recorded on an Agilent 6510 ESI-TOFLC/MS Mass Spectrometer (Agilent, California USA).

Analytical reverse phase HPLC was performed on an Agilent 1100 Series.Protein samples were analysed using an Agilent 6220 ESI-TOF LC/MS MassSpectrometer coupled to an Agilent 1200 LC system (Agilent, Palo Alto,Calif.). All data were acquired and reference mass corrected via adual-spray electrospray ionisation (ESI) source. Acquisition wasperformed using the Agilent Mass Hunter Acquisition software versionB.02.01 (B2116.30). Ionisation mode: Electrospray Ionisation; Drying gasflow: 7 L/min; Nebuliser: 35 psi; Drying gas temperature: 325° C.;Capillary Voltage (Vcap): 4000 V; Fragmentor: 300 V; Skimmer: 65 V; OCTRFV: 250 V; Scan range acquired: 300-3200 m/z Internal Reference ions:Positive Ion Mode=m/z=121.050873 & 922.009798.

Protein desalting and chromatographic separation was performed using anAgilent Poroshell C18 2.1×75 mm, 5 μm column using 5% (v/v) acetonitrileported to waste (0-5 min). Upon desalting of sample the flow was portedback into the ESI source for subsequent gradient elution with (5% (v/v)to 100% (v/v)) acetonitrile/0.1% formic acid over 8 min at 0.25 mL/min.Analysis was performed using Mass Hunter version B.06.00 with BioConfirmsoftware using the maximum entropy protein deconvolution algorithm; massstep 1 Da; Baseline factor 3.00; peak width set to uncertainty.

Size exclusion HPLC was performed on a Shimadzu SCL-10A VP/LC-10 AT VPsystem with a Shimadzu SPD-10A VP UV detector followed by a radiationdetector (Ortec model 276 photomultiplier base with preamplifier, Ortec925-SCINT ACE mate preamplifier, BIAS supply and SCA, Bicron 1M 11/2photomultiplier tube). A Biosuite 125 HR SEC 5 μm 7.8×300 mm column wasused with a flow rate of 0.6 mL/min and Dulbecco's PBS with 5%isopropanol as eluent. Radio-iTLC were analysed using a RaytestRita-Star TLC scanner.

Synthesis of DFO-Squaramide (DFOSq)

A mixture of Desferrioxamine B mesylate (0.20 g, 0.31 mmol) and DIPEA(0.05 mL, 0.3 mmol) was stirred in EtOH (6 mL) at 50° C. After 1 h,3,4-diethoxy-3-cyclobutene-1,2-dione (0.1 mL, 0.7 mmol) in EtOH (9 mL)was added. After a further 30 mins of stirring at 50° C. the solvent wasremoved under reduced pressure, and the residue was triturated with EtOH(3×10 mL). The product was dried in vacuo to give DFOSq as a whitepowder (0.17 g, 83%).

¹H NMR (d₆-DMSO, 500 MHz) δ 9.61 (s, 6H), 8.77 (t, J=5.8 Hz, 1H), 8.58(t, J=5.7 Hz, 1H), 7.77 (d, J=4.7 Hz, 5H), 4.64 (p, J=6.9 Hz, 5H), 3.45(t, J=7.0 Hz, 3H), 3.38 (s, 5H), 3.26 (dd, J=13.0, 6.6 Hz, 1H), 3.00(dd, J=12.7, 6.4 Hz, 10H), 2.56 (d, J=6.5 Hz, 1H), 2.26 (t, J=7.2 Hz,1H), 1.96 (s, 7H), 1.50 (d, J=6.6 Hz, 3H), 1.42-1.31 (m, 3H), 1.24 (ddd,J=20.2, 14.7, 8.0 Hz, 2H); ¹³C NMR (d₆-DMSO, 101 MHz) δ 189.39, 189.30,182.05, 181.84, 176.93, 176.47, 172.58, 172.17, 171.97, 171.30, 170.13,70.18, 68.77, 68.73, 47.09, 47.01, 46.79, 43.68, 43.39, 39.52, 38.42,30.10, 29.90, 29.60, 28.82, 27.56, 26.04, 25.82, 23.50, 22.92, 20.35,15.64; HRMS ESI [M+H⁺]: 685.3768, calculated for (C₃₁H₅₃N₆O₁₁)⁺:685.3767, [M+Na⁺]: 707.3589, calculated for (O₃₁H₅₂NaN₆O₁₁)⁺: 707.3586.

Radiolabelling

Aqueous Na₂CO₃ (2 M, 4.5 μL) was added to a solution of ⁸⁹Zr in 1 Moxalic acid (10 MBq, 10 μL) until the pH increased to 10. HEPES buffer(0.5 M, pH 7, 50 μL) was then added and the solution allowed to standfor 5 min. Neutral pH was confirmed, and then a solution of DFOSq inDMSO (1 μL, 0.18 μmol) was added. After 1 h, reaction completion wasconfirmed by radio-iTLC (Silica infused glass fiber plate, 20 mM pH 5citrate buffer, product R_(f)=0) and SEHPLC (BioSuite 125, 5 μm HR SEC7.8×300 mm column, 20 mM pH 7 Dulbecco's PBS with 5% i-PrOH as eluent,0.6 mL/min, product retention time 21.80 min).

Radio-iTLC

The chromatogram of the control (i.e. no DFOSq) is shown in FIG. 4. Thisshows that the zirconium-containing solution moves with the solventfront (as expected) when DFOSq is not present.

The chromatogram of the ⁸⁹Zr DFOSq complex (60 minutes after addition of⁸⁹Zr) is shown in FIG. 5. This shows that the zirconium has now beenretained on the base line (i.e. the “origin”) by virtue of it beingcomplexed to DFOSq.

Size Exclusion HPLC

The UV-Vis chromatograms (at two different absorption wavelengths of 280and 254 nm) and the radiation chromatogram (obtained using a Geigercounter as the detector) of the control are shown in FIG. 6.

The UV-Vis chromatograms and the radiation chromatogram of the ⁸⁹ZrDFOSq complex (78 hours after addition of ⁸⁹Zr) are shown in FIG. 7.

Synthesis and Analysis of ⁸⁹Zr DFOSq-cRGDfK

A solution of DFOSq (5 mg, 7 μmol) in DMSO (35 μL) was added to cRGDfK(3 mg, 5 μmol) in pH 9 borate buffer (0.5 M, 965 μL). The reactionmixture was allowed to stand at room temperature for 5 d, then purifiedby semi-preperative HPLC (ProteCol C18 column) to DFOSq-cRGDfK as awhite solid (0.002 g, 32%).

HRMS ESI [M+H⁺]: 604.3196, calculated for (C₂₇H₄₂N₉O₇)⁺: 604.3202,[M+2H⁺]: 302.6637, calculated for (C₂₇H₄₃N₉O₇)₂ ⁺: 302.6638.

For radiolabelling, aqueous Na₂CO₃ (2 M, 4.5 μL) was added to a solutionof ⁸⁹Zr in 1 M oxalic acid (10 MBq, 10 μL) until the pH increased to 10.HEPES buffer (0.5 M, pH 7, 50 μL) was then added and the solutionallowed to stand for 5 min. Neutral pH was confirmed, and then asolution of DFOSq-cRGDfK in H₂O (10 μL, 0.008 μmol) was added. After 70min, reaction completion was confirmed by radio-iTLC (Silica infusedglass fiber plate, 0.1 M pH 6 citrate buffer as eluent, productR_(f)=0).

The radio-iTLC chromatogram of ⁸⁹Zr-labelled DFOSq-cRGDfK (taken 60minutes after addition of ⁸⁹Zr) is shown in FIG. 8.

Synthesis and Analysis of ⁸⁹Zr DFOSq-Transferrin

A solution of DFOSq (0.17 mg, 0.25 μmol) in DMSO/H₂O (1:10, 26 μL) wasadded to a solution of human holo-transferrin (1.0 mg, 0.013 μmol) in pH9 borate buffer (0.5 M, 974 μL). The reaction mixture was allowed tostand at room temperature for 17 h, and was then filtered using Amicon10 kDa centrifuge filters. The crude product was washed with NaClsolution (0.9% w/v, 2×400 μL) and the concentrate collected to giveDFOSq-transferrin (1.25 mg, 0.012 μmol). The product was analysed byLCMS (Agilent Poroshell C18 5 μm 2.1 75 mm column), which indicated amixture of transferrin (Tf) with 2-8 chelators, and an average of 4.5chelators/protein (see FIG. 9).

For radiolabelling, aqueous Na₂CO₃ (2 M, 10 μL) was added to a solutionof ⁸⁹Zr in 1 M oxalic acid (1.2 MBq, 20 μL) until the pH increased to10. HEPES buffer (0.5 M, pH 7, 30 μL) was then added and the solutionallowed to stand for 5 min. Neutral pH was confirmed, and then asolution of DFOSq-Tf in 0.9% NaCl (2 μL, 100 μg) was added. After 20min, the reaction was complete and confirmed by radio-iTLC (Silicainfused glass fiber plate, 0.1 M pH 6 citrate buffer as eluent, productRf=0) and SEHPLC (BioSuite 125, 5 μm HR SEC 7.8×300 mm column, 20 mM pH7 Dulbecco's PBS with 5% iPrOH as eluent, product retention time 12.56min).

The radio-iTLC chromatogram of ⁸⁹Zr-labelled DFOSq-Tf (taken 20 minutesafter addition of ⁸⁹Zr) is shown in FIG. 10.

The UV-Vis chromatograms and the radiation chromatogram of ⁸⁹Zr-labelledDFOSq-Tf (20 minutes after addition of ⁸⁹Zr) are shown in FIG. 11.

Synthesis and Analysis of ⁸⁹Zr DFOSq-Herceptin in BT474 Tumour-BearingMice—Study 1

A solution of DFOSq (0.46 mg, 0.67 μmol) in DMSO/H₂O (1:10, 228 μL) wasadded to a solution of clinical grade trastuzumab (5.0 mg, 0.034 μmol),and the reaction mixture was allowed to stand at ambient temperature inpH 9 borate buffer (0.5 M, total volume 1.0 mL). After 16 h, thesolution was concentrated using Amicon 50 kDa centrifuge filters. Thefilter was used to then wash the crude product with NaCl/DMSO solution(0.9% w/v NaCl, 5% DMSO, 4×400 μL) followed by NaCl solution (0.9% w/v,400 μL), and the concentrate was collected to give DFOSq-herceptin (1.4mg, 0.0093 μmol, 28%). The product was analysed by LCMS (AgilentPoroshell C18 5 μm 2.1 75 mm column), which indicated a mixture ofHerceptin (Hero) with 2-7 chelators, and an average of 4.5chelators/antibody (see FIG. 12).

For the radiolabelling, aqueous Na₂CO₃ (2 M, 25 μL) was added to asolution of ⁸⁹Zr in 1 M oxalic acid (55 MBq, 75 μL) until the pHincreased to 10. HEPES buffer (0.5 M, pH 7, 100 μL) was then added andthe solution allowed to stand for 5 min. Neutral pH was confirmed, thena solution of DFOSq-Herc in 0.9% NaCl (4 μL, 225 μg) was added. After 25min, the reaction completion was confirmed by radio-iTLC (Silica infusedglass fiber plate, 0.1 M pH 6 citrate buffer as eluent, productR_(f)=0). The reaction mixture was purified on a PD-10 size exclusioncolumn using pH 7 PBS (20 mM, with 5% sodium gentisate) as eluent. Aftercolumn loading, flow through was discarded and the first fraction (1.5mL, 45 MBq) was collected. The product was analysed by radio-iTLC andSEHPLC (BioSuite 125, 5 μm HR SEC 7.8×300 mm column, 20 mM pH 7Dulbecco's PBS with 5% iPrOH as eluent, product retention time 12.55min).

The radio-iTLC chromatogram of ⁸⁹Zr-labelled DFOSq-Herc (taken 25minutes after addition of ⁸⁹Zr) is shown in FIG. 13.

The radio-iTLC chromatogram of purified ⁸⁹Zr-labelled DFOSq-Herc isshown in FIG. 14.

The UV-Vis chromatograms and the radiation chromatogram of cold (i.e.unlabelled) DFOSq-Herc are shown in FIG. 15.

The UV-Vis chromatograms and the radiation chromatogram of ⁸⁹Zr-labelledDFOSq-Herc (taken 24 hours after purification) are shown in FIG. 16.

Mouse Imaging Using ⁸⁹Zr DFOSq-Herceptin

Two doses of ⁸⁹ZrDFOSq-Herceptin in 20 mM PBS (pH 7) with 5% sodiumgentisate (200 μL, 6.0 MBq each) were prepared and administered to BT474tumour-bearing mice via tail vein injection. PET images were taken atintervals of 22 hours, 46 hours, 94 hours and 8 days post administration(see FIGS. 17 and 18).

The table below (Table 1) sets out the standardized uptake value (SUV)for each mouse at each time point.

TABLE 1 Tumour maximum standardised uptake values (SUVs) for BT474tumour-bearing mice using ⁸⁹Zr DFOSq-herceptin Post administrationtimepoint Mouse 1 SUV_(max) Mouse 2 SUV_(max) 22 h 10.76 11.62 46 h16.17 16.24 94 h 21.97 25.79 8 d 31.73 37.21

A comparative study was also undertaken to compare the efficacy of acompound of the present invention (specifically,⁸⁹Zr(DFO-squarate-trastuzumab)) as a tumour imaging agent, with twoother imaging agents (⁸⁹Zr(DFO-maleimide-trastuzumab) and ⁹ZrCl). The⁸⁹Zr(DFO-maleimide-trastuzumab) was prepared by dissolving a 50-foldexcess of DFO-maleimide in water, and adding it to trastuzumab(Herceptin) in PBS buffer. Unreacted DFO-maleimide was removed by spinfiltration and the purified conjugate was radiolabelled with ⁸⁹Zr(ox)₄as described above for DFO-squarate.

Mice with HER2 positive tumours were injected with two doses of asolution containing either ⁸⁹Zr(DFO-squarate-trastuzumab),⁸⁹Zr(DFO-maleimide-trastuzumab) or ⁸⁹ZrCl, in 20 mM PBS (pH 7) with 5%sodium gentisate (200 μL, 6.0 MBq each). PET images were taken atintervals of 22 hours, 46 hours, 94 hours and 8 days post administrationin respect of the ⁸⁹Zr(DFO-squarate-trastuzumab)-treated mouse, and at24 hours for the ⁸⁹Zr(DFO-maleimide-trastuzumab)- and ⁸⁹ZrCl-treatedmice (see FIGS. 1, 2 and 3, respectively).

Imaging Study Using ⁸⁹Zr DFOSq-Herceptin in BT474 Tumour-BearingMice—Study 2

Trastuzumab (2 mg) was diluted in pH 9.0 borate buffer (0.5 M, 355 μL),and a solution of DFOSq in DMSO (1 mg/mL, 45 μL, 5 eq) was added. Themixture was incubated at ambient temperature for 40 h, and purifiedusing a 50 kDa Amicon spin filter, washing with 4% DMSO in saline (3×300μL), followed by saline only (300 uL). ESI MS analysis of the purifiedconjugate indicated 0-5 chelator attachments, with an average of 2chelators/mAb. The purified DFOSq-trastuzumab solution was usedimmediately for radiolabelling.

⁸⁹Zr Radiolabelling of DFOSq-Trastuzumab

A solution of ⁸⁹Zr in 1 M oxalic acid (150 MBq, 112 μL) was diluted withMilliQ water (250 μL) and aqueous Na₂CO₃ (2 M, 32.5 μL) was added insmall portions until the pH increased to 7. HEPES buffer (0.5 M, pH 7,120 μL) was then added and the solution allowed to stand for 5 min.DFOSq-trastuzumab in 0.9% NaCl (56 μL, 675 μg) was added. After 30 min,reaction completion was confirmed by radio-iTLC (Silica infused glassfiber plate, 0.1 M pH 6 citrate buffer as eluent, product R_(f)=0). Thereaction mixture was purified on a PD-10 size exclusion column using pH7 Dulbecco's PBS (20 mM, with 5% sodium gentisate) as eluent. Aftercolumn loading, flow through was discarded and two fractions (FractionA: 0.5 mL, Fraction B: 1.0 mL, 90.2 MBq) were collected. Fraction B wasanalysed by SE-HPLC (BioSuite 125, 5 μm HR SEC 7.8×300 mm column, 20 mMpH 7 Dulbecco's PBS with 5% iPrOH as eluent, product retention time˜12.5 min).

⁸⁹Zr-DFOSq-Trastuzumab: Mouse PET Imaging (BT474)

Four doses (7.5 MBq each) were drawn up from the purified mAb solutionand administered to BT474 tumour bearing NOD/SCID mice. PET imaging wasperformed at 24, 48 and 96 hours. Mice were harvested at 96 hours forbiodistribution data.

TABLE 2 Tumour maximum standardised uptake values (SUVs) for BT474tumour-bearing mice using ⁸⁹Zr-DFOSq-trastuzumab Mouse ID #3 Mouse ID #5Mouse ID #6 Mouse ID #7 24 h 8.7 11.4 12.2 9.5 48 h 11.6 14.4 12.9 11.096 h 10.8 11.5 9.9 9.1

Synthesis, Analysis and Imaging Study of ⁸⁹Zr DFOSq-Herceptin in SKOV3or LS174T Tumour-Bearing Mice

Conjugation of DFOSq to Trastuzumab

Trastuzumab (10 mg) was diluted in pH 9.0 borate buffer (0.5 M, 1.5 mL),and a solution of DFOSq in DMSO (2 mg/mL, 455 μL, 16 eq) was added. Themixture was incubated at ambient temperature overnight, and purifiedusing a 50 kDa Amicon spin filter, washing with 4% DMSO in saline (2×300μL), followed by saline only (300 uL). ESI MS analysis of the purifiedconjugate indicated 1-6 chelator attachments with an average of 3.4chelators/mAb. The purified DFOSq-trastuzumab solution was stored at 4°C. for 8-9 days prior to radiolabelling.

⁸⁹Zr Radiolabelling of DFOSq-Trastuzumab

A solution of ⁸⁹Zr in 1 M oxalic acid (150 MBq, 195 μL) was diluted withMilliQ water (350 μL) and aqueous Na₂CO₃ (2 M, 65 μL) was added in smallportions until the pH increased to 8. HEPES buffer (0.5 M, pH 7, 200 μL)was then added and the solution allowed to stand for 5 min.DFOSq-trastuzumab in 0.9% NaCl (8 μL, 675 μg) was added. After 1 h,reaction completion was confirmed by radio-iTLC (Silica infused glassfiber plate, 0.1 M pH 6 citrate buffer as eluent, product R_(f)=0). Thereaction mixture was purified on a PD-10 size exclusion column using pH7 Dulbecco's PBS (20 mM, with 5% sodium gentisate) as eluent. Aftercolumn loading, flow through was discarded and the first fraction (1.0mL, 54 MBq) was collected. The product was analysed by SEHPLC (BioSuite125, 5 μm HR SEC 7.8×300 mm column, 20 mM pH 7 Dulbecco's PBS with 5%iPrOH as eluent, product retention time ˜12.5 min).

A solution of ⁸⁹Zr in 1 M oxalic acid (145 MBq, 195 μL) was diluted withMilliQ water (400 μL) and aqueous Na₂CO₃ (2 M, 75 μL) was added in smallportions until the pH increased to 10. HEPES buffer (0.5 M, pH 7, 250μL) was then added and the solution allowed to stand for 5 min.DFOSq-trastuzumab in 0.9% NaCl (8 μL, 675 μg) was added. After 1.5 h,reaction completion was confirmed by radio-iTLC (Silica infused glassfiber plate, 0.1 M pH 6 citrate buffer as eluent, product R_(f)=0). Thereaction mixture was purified on a PD-10 size exclusion column using pH7 Dulbecco's PBS (20 mM, with 5% sodium gentisate) as eluent. Aftercolumn loading, 1 mL of flow through was discarded and the firstfraction (1.0 mL, 47 MBq) was collected. The product was analysed bySEHPLC (BioSuite 125, 5 μm HR SEC 7.8×300 mm column, 20 mM pH 7Dulbecco's PBS with 5% iPrOH as eluent, product retention time ˜12.5min). The signal at 11 mins is presumed to be due to antibodyaggregation as a result of agitation during storage prior toradiolabelling.

⁸⁹Zr-DFOSq-Trastuzumab: Mouse PET Imaging (SKOV3)

Three doses (7.5 MBq each) were drawn up from the purified mAb solutionand administered to SKOV3 tumour bearing NOD/SCID mice. PET imaging wasperformed at 24, 48 and 96 hours. Mice were harvested at 96 hours forbiodistribution data.

TABLE 3 Tumour standardised uptake values (SUVs) for SKOV3tumour-bearing mice using ⁸⁹Zr-DFOSq-trastuzumab Mouse 1 Mouse 2 Mouse 424 h 5.27 6.32 4.68 48 h 6.78 7.37 5.80 96 h 4.87 7.59 4.97

TABLE 4 Biodistribution data of SKOV3 mice using ⁸⁹Zr-DFOSq-trastuzumab.Values are given in % ID/g. Mouse Mouse Mouse Organ ID #1 ID #2 ID #4Mean SD SEM Blood 1.10 1.78 0.47 1.12 0.65 0.33 Lungs 4.17 3.30 1.963.14 1.11 0.56 Heart 1.32 1.67 6.38 3.12 2.83 1.41 Liver 9.26 11.02 9.609.96 0.94 0.47 Kidneys 3.53 4.07 3.14 3.58 0.47 0.23 Muscle 0.55 0.570.34 0.49 0.13 0.06 Spleen 77.40 106.48 118.20 100.69 21.00 10.50 Tumour15.81 18.26 12.37 15.48 2.96 1.48

⁸⁹Zr-DFOSq-Trastuzumab: Mouse PET Imaging (LS174T)

Three doses (7.5 MBq each) were drawn up from the purified mAb solutionand administered to LS174T tumour bearing BALB/c nude mice. PET imagingwas performed at 24, 48 and 96 hours. Mice were harvested at 96 hoursfor biodistribution data.

TABLE 5 Tumour standardised uptake values for LS174T tumour- bearingmice using ⁸⁹Zr-DFOSq-trastuzumab Mouse 5 Mouse 6 Mouse 7 24 h 3.10 3.323.07 48 h 3.70 3.73 4.04 96 h 4.72 5.32 5.65

TABLE 6 Biodistribution data of LS174T mice using⁸⁹Zr-DFOSq-trastuzumab. Values are given in % ID/g. Organ Mouse ID #5Mouse ID #6 Mouse ID #7 Blood 12.82 12.24 11.49 12.18 0.66 0.38 Lungs7.99 7.04 7.38 7.47 0.48 0.28 Heart 4.85 4.41 4.61 4.62 0.22 0.13 Liver6.37 7.88 4.58 6.28 1.65 0.95 Kidneys 6.24 8.78 6.06 7.03 1.52 0.88Muscle 1.34 1.37 1.23 1.31 0.08 0.04 Spleen 17.89 5.53 6.50 9.97 6.883.97 Tumour 13.11 12.47 13.67 13.08 0.60 0.35

Synthesis, Analysis and Imaging Study of ⁸⁹Zr DFO-pH-NCS-Herceptin inSKOV3 Tumour-Bearing Mice

Synthesis of DFOPhNCS

Desferrioxamine (203 mg, 0.309 mmol) was stirred in iPrOH/H₂O (32:3 mL),and a solution of Ph(NCS)₂ (271 mg, 1.41 mmol) in CHCl₃ (20 mL) wasadded. Triethylamine (100 μL, 0.717 mmol) was added immediately, and thereaction mixture was stirred for 1.5 h at ambient temperature. HCl (0.1M, 25 mL) was added and the organic layer was separated. The solvent wasevaporated to give a beige solid which was triturated with CH₂Cl₂. Theremaining solid was filtered off and dried to give DFOPhNCS as a whitepowder (207 mg, 89%). ESIMS [M+H]⁺: 753.34, calculated for(C₃₃H₅₃N₈O₈S₂)⁺: 753.34. Analytical HPLC: Method A, retention time 8.95min. ¹H NMR (500 MHz, dmso) δ 7.98 (s, 1H), 7.78 (s, 2H), 7.57 (d, J=8.8Hz, 2H), 7.36 (d, J=8.9 Hz, 2H), 3.52-3.39 (m, J=13.9, 7.0 Hz, 8H), 3.00(dd, J=12.7, 6.5 Hz, 4H), 2.61-2.54 (m, J=3.9 Hz, 4H), 2.31-2.24 (m,J=10.4, 5.4 Hz, 4H), 1.96 (s, 3H), 1.59-1.45 (m, J=22.1, 14.6, 7.3 Hz,8H), 1.42-1.33 (m, 4H), 1.30-1.16 (m, J=18.8, 15.3, 7.1 Hz, 8H).

Conjugation of DFOPhNCS to Trastuzumab

Procedure followed directly from Vosjan, M. J. W. D.; Perk, L. R.;Visser, G. W. M.; Budde, M.; Jurek, P.; Kiefer, G. E.; van Dongen, G. A.M. S., Nat. Protocols 2010, 5 (4), 739-743.

Trastuzumab (3.03 mg) was diluted in saline (1 mL), and the solutionadjusted to pH 9 with 0.1 M Na₂CO₃. A three-fold excess of DFOPhNCS inDMSO (2.3 mg/mL, 20 μL) was added to the mAb solution in portions withconstant gentle shaking. The mixture was incubated at 37° C. at 550 rpmfor 30 mins, and purified on a PD-10 column using gentisic acid (5mg/mL)/sodium acetate (0.25 M) buffer (pH 5.5) as eluent. The purifiedDFOPhNCS-mAb solution was stored at −20° C. for 5 days prior toradiolabelling. ESI MS analysis of the conjugate indicated 0-1 chelatorattachments, with an average of 0.2 chelators/mAb.

⁸⁹Zr Radiolabelling of DFOPhNCS-Trastuzumab

Procedure followed directly from Vosjan, M. J. W. D. et al (above).

Na₂CO₃ (2 M, 90 μL) was added to a solution of ⁸⁹Zr (200 μL, 55 MBq) inoxalic acid (1 M). The mixture was incubated at ambient temperature forthree minutes with gentle shaking. HEPES buffer (0.5 M, pH 7.2, 300 μL)was then added, followed by the DFOPhNCS-trastuzumab solution (710 μL),then HEPES buffer (0.5 M, pH 7.0, 700 μL). The reaction mixture wasincubated at ambient temperature with frequent gentle shaking. iTLCanalysis (20 mM citric acid, pH 5 as eluent) was performed at varioustimepoints to monitor radiolabelling progress (1 hr: 30% labelling, 1.5hr: 53% labelling, 2 hr: % labelling).

After 2 h reaction time, the mixture was purified using a PD-10 columnconditioned with fresh sodium acetate (0.25 M)/gentisic acid (5 mg/mL)buffer, pH 5-6. The purified ⁸⁹Zr-DFOPhNCS-trastuzumab (1 mL, 21.8 MBq)was analysed by iTLC and SEC-HPLC.

⁸⁹Zr-DFOPhNCS-Trastuzumab: Mouse PET Imaging SKOV3

Three doses (3.5 MBq each) were drawn up from the purified mAb solutionand administered to SKOV3 tumour bearing NOD/SCID mice. PET imaging wasperformed at 24, 48 and 96 hours. Mice were harvested at 96 hours forbiodistribution data.

TABLE 7 Biodistribution data of SKOV3 mice using ⁸⁹Zr-DFOPhNCS-trastuzumab Mouse # Time SUVmax Tmax:Bav Liverav Tmax:Liverav BoneavTmax:Boneav 16 24 3.53 3.41 0.39 1.34 0.17 3.09 48 3.47 4.26 0.37 1.410.18 2.88 96 2.20 4.24 0.41 1.03 0.21 2.02 28 24 5.48 3.96 0.51 1.610.19 4.26 48 5.89 5.12 0.47 1.91 0.18 4.86 96 4.19 6.09 0.42 1.89 0.213.72 33 24 5.30 4.07 0.53 1.48 0.17 4.70 48 4.62 3.71 0.50 1.40 0.174.20 96 3.86 5.36 0.60 1.23 0.22 3.37

Competition Study 1

Synthesis of DFO-cRGDfK Derivatives

An aqueous solution of cRGDfK (100 uL, 2 mg, 2 μmol) was increased to pH9 using sodium carbonate solution. A solution of DFOSq in DMSO (100 uL,2 eq) in pH 9 borate buffer (0.5 M, 100 μL). The reaction mixture wasallowed to stand at room temperature overnight, then purified bysemi-preparative HPLC (ProteCol C18 column, H₂O/MeCN, 0.1% TFA) andfreeze dried to give DFOSq-cRGDfK as a white solid. DFOPhNCS-cRGDfK wasprepared from DFO-Ph-NCS using the same procedure, however due toprecipitation this was centrifuged prior to purification and only thesoluble material was purified. An aqueous solution of each DFO-cRGDfKderivative was prepared at equal concentrations, and this was confirmedby Fe³⁺ titration using UV-Vis spectrometry (425 nm).

Competition Experiment

A solution of ⁸⁹Zr (2 uL, ˜2 MBq) in 1 M oxalic acid was diluted withH₂O (50 uL) and neutralised using 2 M Na₂CO₃ (1 uL), then buffered withpH 7.4 HEPES buffer (5 uL). A small amount of the buffered Zr solution(5 uL) was then added to each DFO-cRGDfK solution (50 uL each). After 20mins, reaction completion was confirmed by radio-iTLC. Each sample wasrun on HPLC (Phenomenex Luna column) as a standard. The DFOPhNCS-cRGDfKligand elutes at 20.1 mins, the Zr complex at 18.5 mins (FIG. 39). TheDFOSq-cRGDfK ligand elutes at 18.0 mins, the Zr complex at 15.5 mins(FIG. 40). An unidentified impurity in the DFOSq-cRGDfK ligand solutionalso eluted at 18.0 minutes, however this did not bind Zr.

A solution of both ligands in equal concentrations (40 uL each) wasprepared and thoroughly mixed, and 5 uL of the buffered Zr solution wasadded. The reaction mixture was analysed after 45 minutes by HPLC, andindicated the formation of exclusively the ZrDFOSq-cRGDfK complex (FIG.41).

Competition Study 2

Synthesis of DFO-503H Derivatives: DFOSqTaur

A small amount of triethylamine (5 drops) was added to a solution oftaurine (18 mg, 0.14 mmol) in H₂O. DFOSq (100 mg, 0.15 mmol) wasdissolved in DMSO and added to the taurine solution, and the reactionmixture stirred at ambient temperature overnight. The solvent wasevaporated and the crude material dissolved in H₂O with gently heating,and any unreacted DFOSq removed by centrifugation. The solvent wasevaporated to give a white powder. FIGS. 42 and 43 show the ¹H NMR andESI-MS spectra of DFOSqTaur.

Synthesis of DFO-503H Derivatives: DFOPhSO₃H

A small amount of triethylamine (5 drops) was added to a solution of4-sulfophenyl isothiocyanate sodium salt monohydrate (38 mg, 0.15 mmol)in MeOH. DFO mesylate (100 mg, 0.15 mmol) was added to the solution, andH₂O (1 mL) was added to improve solubility. The reaction mixture stirredat ambient temperature overnight. The solvent was removed byevaporation, and the crude white powder washed thoroughly with MeOH at40° C. to give the product as a white powder. FIGS. 44 and 45 show the¹H NMR and ESI-MS spectra of DFOPhSO₃H.

Competition Experiment

A 3 mM stock solution of each ligand, DFOPhSO₃H and DFOSqTaur, wasprepared in a mixture of H₂O and MeOH (concentrations were confirmedprior to dilution by UV-Vis Fe³⁺ titration in H₂O, 430 nm). A mixture ofboth solutions (25 uL each) was mixed thoroughly and heated to 50° C. Astock solution of ZrCl₄(THF)₂ in H₂O/MeOH (3 mM) was also prepared, and20 uL of this was added to the ligand mixture. The mixture was incubatedat 50° C. for 7 hours. ESI-MS analysis of the mixture at 7 h (FIG. 46)indicated the presence of the ZrDFOSqTaur complex (FIG. 46), [M]+m/z(calc)=850.22.

1. A conjugate of: a compound of formula (I):

or a pharmaceutically acceptable salt thereof, and a target molecule, wherein L is a leaving group selected from azide, halogen, cyanate and OR, wherein R is selected from C₁ to C₁₀ alkyl, C₁ to C₁₀ heteroalkyl, C₂ to C₁₀ alkene, C₂ to C₁₀ alkyne, and aryl, each of which is optionally substituted.
 2. The conjugate of claim 1, wherein L is OR.
 3. The conjugate of claim 2, wherein R is C₁ to C₆ alkyl.
 4. The conjugate of claim 2, wherein OR is selected from O-p-toluenesulfonate, O-methanesulfonate, O-trifluoromethanesulfonate, O-benzenesulfonate, and O-m-nitrobenzenesulfonate.
 5. The conjugate of claim 1, wherein the target molecule is a polypeptide.
 6. The conjugate of claim 5, wherein the polypeptide is an antibody.
 7. The conjugate of claim 6, wherein the antibody is selected from trastuzumab, rituximab and cetuximab.
 8. The conjugate of claim 1, wherein the target molecule is a peptide.
 9. The conjugate of claim 8, wherein the peptide is a targeting peptide.
 10. The conjugate of claim 9, wherein the targeting peptide is selected from a cyclic RGD sequence, bombesin and glu-N(CO)N-lys PSMA. 